Biochemical characterization of core TFIIH ATPases

Systematic biochemical analysis of core TFIIH ATPases XPB and XPD in the context of NER is currently lacking due to difficulties in obtaining large amounts of recombinant TFIIH and other NER factors. Since we overcame this barrier (Methods), we first measured the ability of XPD and XPB to hydrolyze ATP (Fig. 11a).

Figure 11 | Biochemical characterization of the core TFIIH ATPases.

(a) (left) Raw data of the ATPase assay for the TFIIH core. In the assay ADP recycling is coupled to NADH oxidation which can be monitored in real time by a decline in absorbance at 340 nm (Methods). The assay was performed at different ATP concentrations as indicated next to the plot. (right) The turnover number (kcat) of ATP consumption is shown for the core TFIIH (yellow) and the core TFIIH lacking XPD (gray), in absence of DNA or in the presence of ssDNA or dsDNA as indicated below the plot. Error bars represent the s.d. of the mean values for 2 experimental replicates (b) Polarity of the XPD helicase. DNA unwinding was monitored by a FRET-based assay (Methods). Fluorescence traces of the core TFIIH unwinding in the 5’–3’ and the 3’–

5’ direction are shown in yellow and black, respectively. DNA unwinding of the core TFIIH containing the XPD K48R mutant is shown in gray. Shown traces are representative of 2 independent experiments. (c) Double-stranded DNA translocation by the core TFIIH is mediated by XPB. DNA unwinding and triplex disruption were monitored by a FRET-based assay (Methods). Translocase (left) and helicase (right) activities of the core TFIIH in the presence (yellow) or absence (gray) of triptolide (100 μM final) are shown. Triptolide inhibits XPB^200,201 and specifically affects the core TFIIH translocase activity. Bar graphs show the percentage of disrupted triplex after 4000 s (left) and the percentage of unwound duplex after 300 s (right) for 2

We additionally purified the core TFIIH lacking the XPD subunit, so that the ATP hydrolysis by XPB can be measured directly. An NADH-coupled photometric assay²⁰² was used to monitor ATP hydrolysis in real time (Fig. 11a). Since XPB and XPD are both DNA motor proteins we also tested if the addition of single (ssDNA) or double stranded DNA (dsDNA) will stimulate the ATPase rate of core TFIIH. In the absence of nucleic acids both core TFIIH ATPases are quite inefficient in hydrolyzing ATP (Fig. 11a), probably to prevent futile ATP consumption when their enzymatic function is not required. Upon the addition of excess ssDNA or dsDNA the turnover number of ATP consumption for both heptameric and hexameric core TFIIH assembly increased ~7 times (Fig. 11a). The stimulation of the TFIIH ATPase activity by DNA was also shown before⁷³. However, the contribution of XPB and XPD to ATP hydrolysis is dependent on the type of DNA. XPD is more stimulated by ssDNA while XPB is more stimulated by dsDNA (Fig. 11a), which reflects the different substrate specificities of the two ATPases.

To further investigate XPB and XPD, we established fluorescence resonance energy transfer (FRET)-based assays to monitor their helicase and dsDNA translocase activities in real time. The dsDNA translocase activity (called just translocase activity throughout the thesis) of core TFIIH was measured by a triplex disruption assay, as previously described^69,203. For the helicase activity, we also measured DNA unwinding in 5’-3’ and 3’-5’ direction. Core TFIIH exhibited a robust 3’-5’-3’ helicase activity (Fig. 11b) which could be attributed to XPD as this activity is abolished when XPD is replaced with the XPD point mutant which cannot hydrolyze ATP⁶⁸ (Fig. 11b). This agrees with previous reports which described XPD as a 5’-3’ DNA helicase^72,73. However, core TFIIH did not show any DNA unwinding in 3’-5’ direction (Fig. 11b), the activity which has been demonstrated for archaeal XPB⁶⁷. Core TFIIH also has a translocase activity which can be attributed to XPB because this activity is sensitive to the addition of triptolide, a drug that specifically inactivates XPB^200,201 (Fig. 11c). The translocase activity was also demonstrated for the yeast XPB homologue Ssl2⁶⁹. As a control we showed that triptolide does not influence core TFIIH helicase activity which depends on XPD (Fig. 11c). A comparison of TFIIH enzymatic activities suggests that XPD is the dominant TFIIH motor, since DNA unwinding occurs on a shorter time scale and is more efficient compared to DNA translocation (Fig.

11). In summary, XPD is a 5’-3’ DNA helicase and XPB is a dsDNA translocase.

Next we investigated the effect of NER factors on the core TFIIH ATPases. We purified XPA, RPA heterotrimeric complex, XPG and XPF-ERCC1 complex in large amounts and high purity (Fig. 12a). We first tested the effect of the NER factors on XPD helicase activity. XPD helicase activity was stimulated by both XPA and XPG (Fig. 12b).

Helicase activity was not observed in the presence of RPA complex probably because RPA has a very high affinity for ssDNA^104,204 and masks the DNA overhang XPD uses to initiate DNA unwinding. The XPD helicase activity in the presence of XPG was too fast to be captured with a microplate reader which has a dead time of more than 10 s (Fig. 12b, blue trace).

Figure 12 | Regulation of ATPases in core TFIIH.

(a) Purified XPA, RPA, XPF-ERCC1 and XPG NER factors were resolved by SDS-PAGE and visualized by Coomassie staining. (b) Effect of NER proteins on XPD 5’-3’ helicase activity. Real-time fluorescence measurement using a FRET assay as in Fig. 11b is shown. Bars show percentage of unwound product after 100s. Error bars provide s.d. of the mean of two replicates. (c) (left) Schematic representation of a stopped-flow apparatus. (right) Stopped-stopped-flow measurement of DNA unwinding. TFIIH core (gray) was pre-incubated with excess XPA (purple) or XPG (blue) and rapidly mixed with ATP in a stopped-flow apparatus. DNA unwinding was monitored by a FRET-based assay as in Fig. 11b. Fluorescence traces represent the average of 5 measurements. Initial linear parts of the traces were fitted with Prism software to obtain the initial rate of DNA unwinding, as indicated below the graph. (d) Effect of NER proteins on XPB translocase activity. Real-time fluorescence measurement of triplex disruption as in Fig. 11c is shown. Bars show percentage of disrupted

Therefore, we coupled the helicase assay to a stopped-flow apparatus and resolved the entire DNA unwinding reaction in the presence of XPA and XPG (Fig. 12c). The unwinding was

~4 and ~20 times faster in the presence of XPA and XPG, respectively (Fig. 12c). Strong stimulation of XPD by XPG explains a longstanding observation that XPG is required for efficient repair bubble opening, which is independent of its endonuclease activity¹¹³. We also tested the effect of NER factors on the XPB translocase activity (Fig. 12d). The activity was stimulated by all NER factors tested, however, the stimulation by XPA was exceptionally strong. In the presence of XPA, the percentage of disrupted triplexes increased from ~10% to ~80% after 4000 s (Fig. 12d). Even though the positive effect of XPA on DNA unwinding by TFIIH was known⁵⁶, the stimulation of the XPD helicase and the XPB translocase activity by XPG and XPA, respectively, has not been reported before. This may be important for the lesion scanning step of NER which is mediated by TFIIH migration on the DNA while searching for the lesion⁵⁶.

2.2.2 Core TFIIH-XPA-XPG-DNA complex formation and cryo-EM structure